SUMMARY Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are major causes of respiratory disease and hospitalization in infants. They are also a significant health concern for the elderly, the immunocompromised and individuals who have underlying lung disorders. RSV and HMPV are both members of the family Pneumoviridae, in the order Mononegavirales, the non-segmented negative strand RNA viruses (nsNSVs), and they share their overall scheme of transcription and RNA replication with other viruses with this genome structure. Transcription and replication are both initiated from a promoter at the 3 end of the genome. During transcription, the polymerase moves along the genome and responds to signals at the beginning and end of each gene, yielding subgenomic, capped and polyadenylated mRNAs. During replication, these signals are ignored, and the polymerase generates a full-length complementary antigenome RNA. Studies to investigate how the pneumovirus polymerase is regulated between transcription and replication have found that none of the viral proteins elicits a switch from one process to the other. Instead, the polymerase initiates transcription or replication at two different sites in the promoter (1U for replication, or 3C for transcription) and can become committed to transcription or replication depending on the relative concentrations of the initiating NTPs. How NTPs are distributed in pneumovirus infected cells is not known. Studies of nucleotide biosynthesis pathways have shown that levels and activation of biosynthesis enzymes are tightly regulated and that following certain stimuli, enzymes can reorganize into clusters within the cytoplasm to regulate their activities. The goal of this exploratory project is to examine the relationship between pneumovirus transcription, RNA replication, and nucleotide biosynthesis in infected cells. Aim 1 of the project will focus on determining if RSV and HMPV transcription and replication are regulated temporally over the course of infection, and/or are separated spatially, such that some viral RNA synthesis sites engage in transcription, and others in replication. Aim 2 will focus on characterizing the effect of RSV and HMPV infection on the levels and distribution of NTP biosynthesis enzymes, and the relationship between sites of viral RNA synthesis and areas of ATP and GTP concentration. Integration of the data obtained from Aims 1 and 2 will reveal if the NTP biosynthesis machinery plays a role in regulating pneumovirus transcription and replication. This project brings together a new team with expertise in pneumovirus transcription and replication mechanisms, biochemistry, and cell biology and imaging, and will take advantage of state-of-the-art reagents and techniques for examining viral RNA synthesis and NTP biosynthesis sites at a subcellular level. These studies will provide exciting new insight into the relationship between these important human pathogens and the host cell.